High background and non-specific bands are a particularly annoying and frustrating part of western analysis, particularly when antisera or unpurified antibodies are involved.

Kit Description:

High background and non-specific bands are a particularly annoying and frustrating part of western analysis, particularly when antisera or unpurified antibodies are involved.

GenScript’s Western Optimization Kit offers a quick and complete solution to both these issues. The kit can help you easily find the best blocking reagents for any given antibody (e.g. mTOR polyclonal antibody) in a ninety-minute procedure, eradicating the potential non-specific binding of both primary and secondary antibody to the blocking reagents and unrelated proteins in the samples.

GenScript western optimization technology (patent pending) is currently available for use of rabbit, mouse and goat primary antibody. While the kit’s components are also available piecemeal for tailoring purpose of optimized/customized western blot, every kit can literally optimize ten different antibodies.

Key features:

Optimize Western Conditions for Any Antibody: The kit works with any antibody, even unpurified antibodies and antibodies that usually give high background.

Quick and Simple Ninety-Minute Western Procedure: The kit beats Western blot time down to less than two hours and has fewer and simpler steps than regular Western optimization kits.

No Secondary Antibody Is Needed: The kit adopts our proprietary One-Step Western technology, bypassing the secondary antibody-binding step of the classical Western blot.

Using GenScripts breakthrough immunodetection technology (patent pending), this kit can shorten total western analysis time from the five hours characteristic of the traditional three-step western procedure to just under 90 minutes. Transfer the proteins from gel to membrane and incubate it in the Pretreat Solution for five minutes. Then incubate in WB solution with primary antibody for 40 minutes. Lastly, wash the membrane three times for 10 minutes each. The membrane can then be developed with the HRP substrate included in the kit.

The kit contains all the necessary reagents, buffers, nitrocellulose membrane, and HRP substrate for optimizing western blots. These include four different Pretreat A Solutions (A-a, A-b, A-c, and A-d) for optimization, or one of the four for optimized/customized analysis.

Storage:

Store WestClearTM Nitrocellulose Membrane at room temperature. Store the rest of the kit at 4°C. It will remain stable for six months. Do not freeze the kit or any of its components.

Customized Kits:

The customized kits below each contain one of the four Pretreat Solutions:

Kit Components

10 Assays (Rabbit)

10 Assays (Mouse)

10 Assays (Goat)

Pretreat A-a Solution

L00260

L00264

L00268

Pretreat A-b Solution

L00261

L00265

L00269

Pretreat A-c Solution

L00262

L00266

L00270

Pretreat A-d Solution

L00263

L00267

L00271

Examples:

1. Western blot optimization using unpurified anti-α-tubulin (mouse ascites fluid, Sigma, T 5168) Shown below is a Western blot performed on five strips using unpurified monoclonal anti-α-Tubulin (mouse ascites fluid, Sigma, T 5168). 10 μg of Hela Cell Lysate (BD Bioscience, #611449) was loaded each of the five wells. After gel electrophoresis, proteins were transferred from gel to nitrocellulose membrane (included in the kit). The membrane was cut into five strips with one loaded lane in each strip. One strip was processed following the classic three-step western procedure. The other four strips were processed following optimized procedure from the Western Optimization Kit (L00258). The results are shown in Figure 2.

Figure 2. Western blot for the detection of α-Tubulin in Hela cell lysate following both the classical procedure and optimized procedure from the Western Optimization Kit (L00258) using unpurified mouse antibody. 10 μg of Hela Cell Lysate (BD Bioscience, #611449) was loaded into each of the five wells. The blots were developed with the LumiSensorTM Plus Chemiluminescent HRP Substrate Kit included in the kit. From the results, it can be seen that Pretreat A-c gave the best results. α-Tubulin is detected without any background or non-specific bands. 2. Western blot optimization using Rabbit Anti-mTOR (Phospho-Ser2448) pAb (Cell Signaling Technology, #2972) Shown below is a Western blot performed using five Western blot strips and purified Rabbit Anti-mTOR (Phospho-Ser2448) pAb (Cell Signaling Technology, #2972) to detect mammalian target rapamycin, mTOR. 10 μg of Hela Cell Lysate (BD Bioscience, #611449) was loaded into each of the five wells. After gel electrophoresis, the proteins were transferred from gel to the nitrocellulose membrane (included in the kit). The membrane was cut into five strips with one loaded lane in each strip. One strip was processed following the classical three-step Western procedure. The other four strips were processed following the optimized procedure from the Western Optimization Kit (L00257). In accordance with the manufacture’s recommendation, the antibody incubation was performed overnight on a shaker at 4°C. The results are shown in Figure 3.

Figure 3. Western blot for the detection of mTOR in Hela cell lysate following both the classical procedure and the optimization procedure from the Western Optimization Kit (L00257) using Rabbit Anti-mTOR (Phospho-Ser2448) pAb. 10 μg of Hela Cell Lysate (BD Bioscience, #611449) was loaded into each of the five wells. The blots were developed with the LumiSensorTM Plus Chemiluminescent HRP Substrate Kit included in the kit.